Core-Shell Structured PLGA Particles Having Highly Controllable Ketoprofen Drug Release

The non-steroid anti-inflammatory drug ketoprofen (KP) as a model molecule is encapsulated in different poly(lactide-co-glycolide) (PLGA) nanostructured particles, using Tween20 (TWEEN) and Pluronic F127 (PLUR) as stabilizers to demonstrate the design of a biocompatible colloidal carrier particles with highly controllable drug release feature. Based on TEM images the formation of well-defined core-shell structure is highly favorable using nanoprecipitation method. Stabile polymer-based colloids with ~200–210 nm hydrodynamic diameter can be formed by successful optimization of the KP concentration with the right choice of stabilizer. Encapsulation efficiency (EE%) of 14–18% can be achieved. We clearly confirmed that the molecular weight of the stabilizer thus its structure greatly controls the drug release from the PLGA carrier particles. It can be determined that ~20% and ~70% retention is available with the use of PLUR and TWEEN, respectively. This measurable difference can be explained by the fact that the non-ionic PLUR polymer provides a steric stabilization of the carrier particles in the form of a loose shell, while the adsorption of the non-ionic biocompatible TWEEN surfactant results in a more compact and well-ordered shell around the PLGA particles. In addition, the release property can be further tuned by decreasing the hydrophilicity of PLGA by changing the monomer ratio in the range of ~20–60% (PLUR) and 70–90% (TWEEN).


Preparation of the Drug-Free and KP-Loaded PLGA NPs
For the preparation of the PLGA50 drug carriers, nanoprecipitation method was used under different initial parameters: c PLUR/TWEEN = 0.0-1.0 mg/mL, c PLGA = 2.5-10.0 mg/mL, c NaCl = 0.0-0.16 M. During the formation of the drug-free particles, the PLGA50 was dissolved in organic solvent (acetone, or DMSO), and 0.5 mL stock solution was added dropwise to 5 mL stabilizer (PLUR or TWEEN)-contained aqueous phase (organic phase to aqueous phase ratio 1:10). The samples were stirred at 350 rpm at room temperature for a day and after that the prepared PLGA50 particles were stored at 10 • C until the further used.
In case of the KP-loaded PLGA systems, firstly the copolymer derivatives (PLGA50/65/75) (c PLGA = 10 mg/mL) were dissolved in acetone making individual samples which also contained different concentrations of dissolved KP (c KP = 0-15 mg/mL). Secondly, 0.5 mL of this organic phase was dropped to an aqueous TWEEN or PLUR stabilizer-containing solution (V = 5 mL, c = 0.1 mg/mL) which results in the precipitation of PLGA particles. To eliminate the nonencapsulated KP molecules the dispersions were centrifuged (12,000 rpm, 15-20 min) and the supernatant was removed. Dispersions were freeze-dried (using Christ Alpha 1-2 LD plus equipment) to obtain solid samples for further structural characterizations. The solid samples were stored at −20 • C.

Methods for Size, Size Distribution and Structural Characterizations
The transmission electron microscopy (TEM) images were recorded by Jeol JEM-1400plus equipment (JEOL Ltd., Tokyo, Japan) using 120 keV accelerating voltage. The particle size, size distribution and the ζ-potential values of the drug-free PLGA and KPloaded PLGA particles were determined with a Horiba Sz-100 (HORIBA Jobin Yvon, Longjumeau, France) dynamic light scattering (DLS) equipment, which has a diode pumped frequency doubled (532 nm, 10 mW) laser. For evaluation of the DLS data both the refractive indexes and the viscosities of the solvent mixtures were determined. The parallel experiments were performed with 90 • of detection angle at 25 ± 0.1 • C. To determine the encapsulation efficiency (EE%) and the drug loading (DL%) of the carriers, Shimadzu UV-1800 UV-Vis double beam spectrophotometer was used. The lyophilized drug-loaded carriers were dissolved in 1,4-dioxane and sonicated for 30 s. The UV-Vis spectra of the samples were registered in the range of 200-400 nm using 1 cm quartz cuvette at room temperature, and the concentration of the KP was calculated from the calibration curve ( Figure S1). After the purification of the samples the amount of the non-encapsulated drug molecules in the supernatant was also determined by this way. The characteristic absorbance band was detected at 252 nm (in 1-4-dioxane). The EE% (Equation (1)) and the DL% (Equation (2)) were determined by the initial drug mass used in the formulation and the total mass of the NPs, respectively. EE% = encapsulation mass of drug total mass of drug × 100 (1) DL% = encapsulation mass of drug total mass of nanoparticles × 100 Differential scanning calorimetry (DSC) curves of the solid samples were recorded by Mettler Toledo DSC822e in the temperature range of 25-500 • C, where nitrogen with 50 mL/min flow rate was used. The heating rate was 5 • C/min in every cases. FT-IR spectra of the KP-loaded PLGA systems were obtained by a Jasco FT/IR-4700 with ATR PRO ONE Single-reflection accessory (ABL&E-JASCO, Budapest, Hungary) at room temperature. The measurements were carried out in 500-4000 cm −1 wavenumber range with 1 cm −1 resolution at room temperature. The spectra were determined by 128 interferograms.

In Vitro Release Studies
The in vitro release curves were obtained by UV-Vis spectrophotometer, where the dissolution profiles were registered at different pH (pH = 6, 7.4 and 8; phosphate buffer with 0.9% NaCl) and temperatures (T = 25, 37 and 45 • C). The quantity of the active substance released was determined by calibration curves at λ = 260 nm (in PBS medium) ( Figure S2). The pure drug and the drug-contained samples were placed separately in a cellulose (dialysis) membrane (M w cut-off = 14,000 Da; Sigma-Aldrich) with 5 mL buffer solution, which were inserted into 35 mL dissolution medium, and the penetration of the KP molecules was followed for 420 min. The dissolution curves were fitted by several kinetic equations (first order, second order, Higuchi, Weibull and Korsmeyer-Peppas models) using nonlinear regression [31,32] to provide more information on the release mechanism.

Influence of the Initial Synthesis Conditions on the Drug-Free PLGA50 Particles
The size, size distribution and the structure of the PLGA50-based particles can be tuned with the optimization of the preparation parameters. To highly control the hydrodynamic diameter of the formed particles the fabrication of the PLGA drug carrier particles was carried out by using different organic solvents (acetone or DMSO), two types of stabilizers (PLUR or TWEEN) and various polymer concentrations. The measured diameters with the polydispersity indexes determined by DLS are shown in Figures 1 and S3. The measurements were carried out in 500-4000 cm −1 wavenumber range with 1 cm −1 resolution at room temperature. The spectra were determined by 128 interferograms.

In Vitro Release Studies
The in vitro release curves were obtained by UV-Vis spectrophotometer, where the dissolution profiles were registered at different pH (pH = 6, 7.4 and 8; phosphate buffer with 0.9% NaCl) and temperatures (T = 25, 37 and 45 °C). The quantity of the active substance released was determined by calibration curves at λ = 260 nm (in PBS medium) (Figuew S2). The pure drug and the drug-contained samples were placed separately in a cellulose (dialysis) membrane (Mw cut-off = 14,000 Da; Sigma-Aldrich) with 5 mL buffer solution, which were inserted into 35 mL dissolution medium, and the penetration of the KP molecules was followed for 420 min. The dissolution curves were fitted by several kinetic equations (first order, second order, Higuchi, Weibull and Korsmeyer-Peppas models) using nonlinear regression [31,32] to provide more information on the release mechanism.

Influence of the Initial Synthesis Conditions on the Drug-Free PLGA50 Particles
The size, size distribution and the structure of the PLGA50-based particles can be tuned with the optimization of the preparation parameters. To highly control the hydrodynamic diameter of the formed particles the fabrication of the PLGA drug carrier particles was carried out by using different organic solvents (acetone or DMSO), two types of stabilizers (PLUR or TWEEN) and various polymer concentrations. The measured diameters with the polydispersity indexes determined by DLS are shown in Figures 1 and S3. The results of the concentration-dependence of the two stabilizers, where the amount of the PLGA was constant (cPLGA = 5 mg/mL), demonstrate that the particle sizes are not The results of the concentration-dependence of the two stabilizers, where the amount of the PLGA was constant (c PLGA = 5 mg/mL), demonstrate that the particle sizes are not changed significantly with their increasing concentrations when the acetone is used as organic phase ( Figure 1A,B). In contrast, slightly increasing tendency in the diameters can be seen for use of DMSO. Moreover, we can clearly state that the application of DMSO results in significantly lower values (d~60-80 nm) than it was observed for acetone (d~110-120 nm). These experimental results highly indicate that the quality of the organic phase has an important effect on the formation of PLGA50 carrier particles.
Based on the results presented on Figure 1A,B, 0.1 mg/mL TWEEN and PLUR stabilizer concentration was applied for further studies. After optimization of the stabilizer concentration, the effect of the concentration of the PLGA50 macromolecules was also studied on the particle formation ( Figure 1C,D). It can be concluded that dominantly higher hydrodynamic diameter values can be measured by increasing of the PLGA amount from 2.5 mg/mL to 15 mg/mL, independently from the type of the stabilizers and the organic solvents. The values of the particle size are varied in the range of~100-160 nm for PLUR and between~100-150 nm in case of TWEEN in acetone. The~10 nm difference between the two stabilizers is presumably due to their different molecular weights (M w,TWEEN = 1228 Da, M w,PLUR =~12,600 Da). In case of the DMSO-containing systems, similar tendency is observed, but in a smaller particle size range (~60-95 nm for PLUR and~50-90 nm for TWEEN). The polydispersity indexes ( Figure S3) are not changed measurable.
To characterize their colloid stability, the effect of NaCl concentration (c NaCl = 0-0.16 M) was also studied on the change of the particle size in the absence and in the presence of the stabilizers. The Figure 2 shows that the increasing of the NaCl concentration results larger particle size and polydispersity, while the ζ-potential values are decreased. changed significantly with their increasing concentrations when the acetone is used as organic phase ( Figure 1A,B). In contrast, slightly increasing tendency in the diameters can be seen for use of DMSO. Moreover, we can clearly state that the application of DMSO results in significantly lower values (d ~60-80 nm) than it was observed for acetone (d ~110-120 nm). These experimental results highly indicate that the quality of the organic phase has an important effect on the formation of PLGA50 carrier particles. Based on the results presented on Figure 1A,B, 0.1 mg/mL TWEEN and PLUR stabilizer concentration was applied for further studies. After optimization of the stabilizer concentration, the effect of the concentration of the PLGA50 macromolecules was also studied on the particle formation ( Figure 1C,D). It can be concluded that dominantly higher hydrodynamic diameter values can be measured by increasing of the PLGA amount from 2.5 mg/mL to 15 mg/mL, independently from the type of the stabilizers and the organic solvents. The values of the particle size are varied in the range of ~100-160 nm for PLUR and between ~ 100-150 nm in case of TWEEN in acetone. The ~10 nm difference between the two stabilizers is presumably due to their different molecular weights (Mw,TWEEN = 1228 Da, Mw,PLUR = ~12,600 Da). In case of the DMSO-containing systems, similar tendency is observed, but in a smaller particle size range (~ 60-95 nm for PLUR and ~ 50-90 nm for TWEEN). The polydispersity indexes ( Figure S3) are not changed measurable.
To characterize their colloid stability, the effect of NaCl concentration (cNaCl = 0-0.16 M) was also studied on the change of the particle size in the absence and in the presence of the stabilizers. The Figure 2 shows that the increasing of the NaCl concentration results larger particle size and polydispersity, while the ζ-potential values are decreased. The aggregation of the particles can be observed at higher amount of NaCl. This observation clearly proves that the PLGA50 carrier particles, without the use of stabilizers, have an electrostatic stability, and due to this, it has a significant sensitivity to the change The aggregation of the particles can be observed at higher amount of NaCl. This observation clearly proves that the PLGA50 carrier particles, without the use of stabilizers, have an electrostatic stability, and due to this, it has a significant sensitivity to the change of the ionic strength. We also confirmed that, higher aggregation tendency can be observed in case of the application of DMSO. After the characterization of the stabilizer-free PLGA50 NPs, we also determined the NaCl effect in presence of TWEEN ( Figure 3) and PLUR ( Figure S4) stabilizers (c stabilizer = 0.1 mg/mL). Na -ions, thus the electrostatic stability disappears in the presence of the inert salt, which causes the increasing of the particle size. However, the TWEEN provides steric stability to the systems, thus preventing the further aggregation. The adsorption of the surfactant is more favored to a near-neutral surface than on a surface that is denser in charge. Similar changes can be seen at the PLUR: dDLS = 116 nm, ζ = −31 mV (cNaCl = 0 M) and dDLS = 219 nm, ζ = 2.5 mV (cNaCl = 0.16 M) with acetone organic phase; dDLS = 70 nm, ζ = −29 mV (cNaCl = 0 M) and dDLS = 251 nm, ζ = −1.2 mV with DMSO organic phase ( Figure S4).

Characterization of the KP-Loaded PLGA NPs
Considering the previous results presented in Section 3.1, the following PLGA50 and stabilizer concentrations were used for encapsulation of KP as a model active compound: cPLGA50 = 10 mg/mL; VPLGA50 = 0.5 mL; cPLUR/TWEEN = 0.1 mg/mL; VPLUR/TWEEN = 5 mL. The increasing of the particle size was lower in the presence of the NaCl, when acetone was used, thus we applied this organic solvent for preparation of PLGA50/KP NPs. Particles were fabricated at different KP concentrations (cKP = 0-15 mg/mL) and the size, size distribution, encapsulation efficiency (EE%) and the drug loading (DL%) were determined in every cases. The results can be seen in Table 1. The yield of the solid carrier NPs was 60-70% in every case, compared to the initial component mass. Particle size of the PLUR-and TWEEN-stabilized PLGA50/KP NPs is increased with KP concentration confirming the In the TWEEN/PLGA50 systems, the hydrodynamic diameter is slightly increased with the NaCl concentration to a constant value (in acetone: d DLS~2 20 nm; in DMSO: d DLS~2 55 nm), where the dominant aggregation of the carriers cannot be detected (Figure 3). Due to the added NaCl, the ζ-potential is changed from −27 mV (c NaCl = 0 M (in acetone) and −23 mV (c NaCl = 0 M in DMSO) to~0 mV, independently from the organic phase. Most probably the negatively charged carboxylate groups of PLGA50 rapidly compensate with Na + -ions, thus the electrostatic stability disappears in the presence of the inert salt, which causes the increasing of the particle size. However, the TWEEN provides steric stability to the systems, thus preventing the further aggregation. The adsorption of the surfactant is more favored to a near-neutral surface than on a surface that is denser in charge. Similar changes can be seen at the PLUR: d DLS = 116 nm, ζ = −31 mV (c NaCl = 0 M) and d DLS = 219 nm, ζ = 2.5 mV (c NaCl = 0.16 M) with acetone organic phase; d DLS = 70 nm, ζ = −29 mV (c NaCl = 0 M) and d DLS = 251 nm, ζ = −1.2 mV with DMSO organic phase ( Figure S4).

Characterization of the KP-Loaded PLGA NPs
Considering the previous results presented in Section 3.1, the following PLGA50 and stabilizer concentrations were used for encapsulation of KP as a model active compound: c PLGA50 = 10 mg/mL; V PLGA50 = 0.5 mL; c PLUR/TWEEN = 0.1 mg/mL; V PLUR/TWEEN = 5 mL. The increasing of the particle size was lower in the presence of the NaCl, when acetone was used, thus we applied this organic solvent for preparation of PLGA50/KP NPs. Particles were fabricated at different KP concentrations (c KP = 0-15 mg/mL) and the size, size distribution, encapsulation efficiency (EE%) and the drug loading (DL%) were determined in every cases. The results can be seen in Table 1. The yield of the solid carrier NPs was 60-70% in every case, compared to the initial component mass. Particle size of the PLURand TWEEN-stabilized PLGA50/KP NPs is increased with KP concentration confirming the successful encapsulation of the drug. The EE% and the DL% are also increased in presence of PLUR, while maximum DL% is reached at c KP = 10 mg/mL (EE%~61.9; DL%~38.2) when TWEEN is used as stabilizer. In addition, we found that the amount of the encapsulated KP cannot be changed indefinitely with the drug concentration, because the particles aggregate after few days above a given concentration (c KP = 5 mg/mL) ( Figure S5). To determine the stability of the drug-free and KP-loaded carriers in detail, the hydrodynamic diameter of the samples was monitored for 25 days by DLS. The data are summarized in Figure S5. The particle size is changed slightly to the lower values, probably, due to the degradation as well as the hydrolysis of the carriers and the decreasing of the KP content. To follow the kinetic of the decreasing of the hydrodynamic diameters, the data were fitted by zero order formulation. The determined rate constants are k = 0.884 nm/h (0 mg/mL KP) and k = 1.523 nm/day (5 mg/mL KP) for PLUR and k = 0.862 nm/day (0 mg/mL KP) and k = 1.017 nm/day (5 mg/mL KP) for TWEEN. Using these, the KP was also encapsulated in different PLGA (PLGA50; PLGA65 and PLGA75) at c KP = 5 mg/mL, where similar data was observed, independently to the lactide:glycolide ratio and the applied stabilizer (Table S1). The morphology of the drug-free and a KP-loaded PLGA50 NPs with TWEEN and PLUR was characterized by TEM. The registered images in Figure 4. show that spherical structure can be identified in the absence of KP. However, it can be concluded, formation of the core-shell structure is highly confirmed in the presence of KP drug, where the stabilizers are formed shell on the KP-contained PLGA core ( Figure 4C,D). Thinner layer can be observed in case of the TWEEN 20, presumably due to the lower molecular weight. This proves that the core-shell structure can be appeared with hydrophobic drugs. The average diameters measured by TEM image are in good agreement with DLS results.
The structure of the drug-loaded particles was studied by FT-IR spectroscopy and DSC measurements. Shift in the IR-spectra of the KP-loaded PLGA50 cannot be observed compared to the spectrum of the pure PLGA50 samples, which suggests that the formation of strong interaction is not realized, therefore its role is negligible during the dissolution of the active compound ( Figure 5A,B).
The appearance of the stretching vibration of the C=O (ν = 1695 cm −1 ) and the keto (ν = 1655 cm −1 ) on the spectra (gray dotted line), moreover the 715 cm −1 , 703 cm −1 and 690 cm −1 bands of deformation vibration of the KP groups in the fingerprint region (ν ≤ 1500 cm −1 ) confirm the presence of encapsulated drug [33][34][35]. Similar changes can be detected in case of the PLGA65 and 75 ( Figure S6). Based on the DSC curves, the endotherm melting point of the crystallic KP is 88 • C, and the degradation can be seen in 300-400 • C temperature range with 377 • C degradation peak maximum (T d,max ) ( Figure 5C,D) [36]. The endothermic T d,max of the PLGA50 is decreased from 357 • C to 349 • C (PLUR/PLGA50/KP) and 347 • C (TWEEN/PLGA50/KP) with the decreasing of the KP concentration, from which the presence of a weak hydrophobic interaction can be assumed.  The structure of the drug-loaded particles was studied by FT-IR spectroscopy and DSC measurements. Shift in the IR-spectra of the KP-loaded PLGA50 cannot be observed compared to the spectrum of the pure PLGA50 samples, which suggests that the formation of strong interaction is not realized, therefore its role is negligible during the dissolution of the active compound ( Figure 5A,B).   The structure of the drug-loaded particles was studied by FT-IR spectroscopy and DSC measurements. Shift in the IR-spectra of the KP-loaded PLGA50 cannot be observed compared to the spectrum of the pure PLGA50 samples, which suggests that the formation of strong interaction is not realized, therefore its role is negligible during the dissolution of the active compound ( Figure 5A,B).

In Vitro Drug Release Study
In case of well-defined core-shell structure, the retention can be tuned with thickness and compactness of the shell, in contrast to the homogenous distribution of the carrier components, where only the polymer matrix influences the dissolution of the encapsulated drugs. Taking this into account, the dissolution processes were followed to investigate the release properties of the core-shell structured TWEEN/PLGA/KP and PLUR/PLGA/KP carriers. During the studies, the measurement conditions as the pH of the phosphate buffer (pH = 6-8), the temperature (T = 25-45 • C) and the monomer ratios of the PLGA (50:50, 65:35 and 75:25 lactide:glycolide ratio) were systematically changed (Figures 6, 7 and S7). The reference was the solid KP in every cases. The temperature-dependence examinations point out that increase in the temperature results in higher drug release which is in line with increasing solubility. Moreover, we can establish that the PLUR/PLGA50/KP particles ( Figure 6A) have lower retention than the TWEEN/PLGA50/KP systems ( Figure 6B), which is based on the fact that the TWEEN molecules having relatively lower molecular weight form a more compact adsorption layer around the PLGA particles than the PLUR with larger M w thus the drug dissolution is more regulated.
In case of well-defined core-shell structure, the retention can be tuned with thickness and compactness of the shell, in contrast to the homogenous distribution of the carrier components, where only the polymer matrix influences the dissolution of the encapsulated drugs. Taking this into account, the dissolution processes were followed to investigate the release properties of the core-shell structured TWEEN/PLGA/KP and PLUR/PLGA/KP carriers. During the studies, the measurement conditions as the pH of the phosphate buffer (pH = 6-8), the temperature (T = 25-45 °C) and the monomer ratios of the PLGA (50:50, 65:35 and 75:25 lactide:glycolide ratio) were systematically changed (Figures 6, 7 and S7). The reference was the solid KP in every cases. The temperaturedependence examinations point out that increase in the temperature results in higher drug release which is in line with increasing solubility. Moreover, we can establish that the PLUR/PLGA50/KP particles ( Figure 6A) have lower retention than the TWEEN/PLGA50/KP systems ( Figure 6B), which is based on the fact that the TWEEN molecules having relatively lower molecular weight form a more compact adsorption layer around the PLGA particles than the PLUR with larger Mw thus the drug dissolution is more regulated.  To analyze the contribution of the carriers to the retention of the drug, the values of the ct/c∞ of the PLUR/PLGA50/KP NPs and TWEEN/PLGA50/KP NPs at 420 min were subtracted from the solid KP values. The Figure 6D. shows, while large effect cannot be seen in TWEEN/PLGA50/KP NPs, until then the retention of the PLUR/PLGA50/KP NPs is reduced from 12.7% (25 °C) to 6.0% (45 °C). Based on these, it can be concluded, the PLUR-stabilized carriers have temperature-sensitive release properties, presumably thanks to the surfactant. Significant change in the release profiles cannot be observed at the pH-dependence measurements, where the retention of the PLUR/PLGA50/KP NPs and the TWEEN/PLGA50/KP NPs are ~ 20% and ~ 70%, respectively ( Figure S7). It is confirmed, the pH shift is not influenced the dissolution of the KP.
To confirm the dominant effect of the monomer ratio in the copolymer, the KP release from PLGA50-, PLGA65-and PLGA75-based colloidal carriers was also studied at pH = 7.4 in PBS medium at 37 °C to model physiological conditions. According to the results, which are shown in Figure 7, the amount of the liberated KP is increased for both the PLUR (ct/c∞ (%) = 77.8 (PLGA50), ct/c∞ (%) = 83.0 (PLGA65) and ct/c∞ (%) = 89.5 (PLGA75)) and the TWEEN (ct/c∞ (%) = 19.6 (PLGA50), ct/c∞ (%) = 33.3 (PLGA65) and ct/c∞ (%) = 62.2 (PLGA75)) stabilizers. Similar to our previous research work on the (±)-α tocopherol (TP)-loaded PLGA NPs, the higher lactide content promotes the release of the drug into the buffer medium [30]. However, the encapsulation efficiency of the KP is not changed systematically with the monomer ratios in contrast to the TP-contained systems, where this effect can be influenced the dissolution processes. Therefore, these results are clearly indicated that ex- To analyze the contribution of the carriers to the retention of the drug, the values of the c t /c ∞ of the PLUR/PLGA50/KP NPs and TWEEN/PLGA50/KP NPs at 420 min were subtracted from the solid KP values. The Figure 6D. shows, while large effect cannot be seen in TWEEN/PLGA50/KP NPs, until then the retention of the PLUR/PLGA50/KP NPs is reduced from 12.7% (25 • C) to 6.0% (45 • C). Based on these, it can be concluded, the PLUR-stabilized carriers have temperature-sensitive release properties, presumably thanks to the surfactant. Significant change in the release profiles cannot be observed at the pH-dependence measurements, where the retention of the PLUR/PLGA50/KP NPs and the TWEEN/PLGA50/KP NPs are~20% and~70%, respectively ( Figure S7). It is confirmed, the pH shift is not influenced the dissolution of the KP.
Similar to our previous research work on the (±)-α tocopherol (TP)-loaded PLGA NPs, the higher lactide content promotes the release of the drug into the buffer medium [30]. However, the encapsulation efficiency of the KP is not changed systematically with the monomer ratios in contrast to the TP-contained systems, where this effect can be influenced the dissolution processes. Therefore, these results are clearly indicated that exclusively the lactide to glycolide ratios have role in the drug retention. The release rate is higher at the TWEEN/PLGA/KP NPs, compared to the PLUR-stabilized systems, thus the influence of the lactide content can be regulated by the optimalization of the surfactants.
For the determination of the kinetic parameters, the release curves of the KP were fitted by different kinetic equations (First order, second order, Weibull, Higuchi and Korsmeyer-Peppas formulations) with nonlinear regression The Weibull and the Second order models gave the best fit at the PLUR/PLGA/KP NPs, while the Weibull and the Korsmeyer-Peppas kinetic formulations showed the best results for TWEEN/PLGA/KP NPs. The appropriate kinetic data are shown in Table S2. The diffusion-dissolution index (n) from Korsmeyer-Peppas model can be determined the erosion and diffusion contribution to dissolution processes (n = 0.42 (diffusion-controlled mechanism), n = 1 (Case II relaxationcontrolled mechanism) and 0.42 ≤ n ≤ 1 (both of them) in case of the sphere shape). At the PLUR/PLGA/KP NPs, the diffusion dissolution index is n = 0.2503 (PLGA50), n = 0.2811 (PLGA65) and n = 0.2397 (PLGA75), which low values can be explained with the polydispersity of the particles [37]. With this in mind, it is confirmed that the KP release from the carriers has diffusion-controlled feature. The TWEEN/PLGA/KP NPs have higher n values, thus the appearance of the relaxation-controlled mechanism is proved. This can be presumably related to the erosion of the TWEEN layer, which is confirmed by the difference between the release profiles of the TWEEN-and PLUR-stabilized PLGA carriers.

Conclusions
In our work, we clearly highlighted the effect of the main preparation conditions (like component ratios, NaCl concentration or type of the organic phase and the stabilizer structure) on the size, structure, the encapsulation efficiency, and the controlled drug release of KP-loaded PLGA particles. Considering the DLS measurements, we can be stated that the preparation of the PLGA carriers is a designable process, where the hydrodynamic diameter of the particles can be systematically changed by the concentration of the polymers and the stabilizers. It can be successfully concluded that~50 nm smaller particle size can be achieved with using DMSO instead of acetone using the nanoprecipitation protocol.
The stability experiments highlighted that, the stabilizer-free PLGA NPs have electrostatic stability, and it disappears at 0.02 M NaCl concentrations. However, due to the steric stability, this effect can be controlled with the use of non-ionic PLUR and TWEEN surfactants having non-toxic features. During the encapsulation of the KP, it can be confirmed, that the formation of the core-shell structured KP-loaded PLGA NPs is possible by the nanoprecipitation method. The optimal initial drug concentration was determined at 5 mg/mL with 7-8% drug loading, because the aggregation of the systems can be observed at higher concentration. The time-dependence measurements of particle size for the drug-free and the 5 mg/mL KP-loaded PLGA50 NPs pointed out that the diameters are continuously decreased, presumably, due to the degradation of the carriers. The determined in vitro release profiles indicate that~60% higher retention can be achieved in case of the TWEEN, regardless of the pH and the temperature of the medium. We highlighted that the type of the stabilizer has more influence on the dissolution processes. Namely, the TWEEN molecules having relatively lower molecular weight form a more compact adsorption layer around the PLGA particles than the PLUR with larger M w thus the drug dissolution is more regulated.
The release curves were fitted by different kinetic models, where the value of the diffusion -dissolution index (n) from the Korsmeyer-Peppas formulation is pointed out that the erosion-controlled features in addition to the diffusion-controlled mechanism are also present in the TWEEN-stabilized systems, while the release of the KP from the PLUR/PLGA/KP NPs only has diffusion properties.